Structural studies and mechanism of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase: insights into the initial step of synthesis of dolichyl-phosphate-linked oligosaccharide chains in membranes of endoplasmic reticulum

• Published in: Glycobiology (Oxford) Vol. 16; no. 7; pp. 666 - 678
• Main Authors: Lamani, Ejvis, Mewbourne, R. Brandon, Fletcher, Damona S, Maltsev, Sergei D, Danilov, Leonid L, Veselovsky, Vladimir V, Lozanova, Antonina V, Grigorieva, Natalia Ya, Pinsker, Olga A, Xing, Jun, Forsee, W. Thomas, Cheung, Herbert C, Schutzbach, John S, Shibaev, Vladimir N, Jedrzejas, Mark J
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.07.2006; Oxford Publishing Limited (England)
• Subjects: (5-iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid; 4-dimethylaminophenylazophenyl-4′-maleimide; Amino Acid Sequence; Binding Sites; Catalysis; DAMBI; deoxthymidine 5′-diphosphate; dithiothreitol; Dol-P; Dol-P mannose; Dol-P-Man; Dolichol Monophosphate Mannose - metabolism; Dolichol Phosphates - metabolism; dolichyl-P; dolichyl-phosphate; dTDP; DTT; endoplasmic reticulum; Endoplasmic Reticulum - enzymology; fluorescence resonance energy transfer; Fluorescent Dyes - chemistry; FRET; GDP; GDP-mannose; glycosyltransferase; GT-2; guanosine 5′-diphosphate; IAEDANS; Intracellular Membranes - enzymology; Mannosyltransferases - chemistry; membranes; modeling; Models, Molecular; molecular mechanism of action; Molecular Sequence Data; Nonidet P40 (ethylphenylpolyethyleneglycol); NP-40; Oligosaccharides - biosynthesis; Protein Conformation; protein glycosylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - chemistry; Spectroscopy, Fourier Transform Infrared; spore coat polysaccharide biosynthesis protein A; SpsA; Substrate Specificity; Tris; tris (hydroxymethyl)aminomethane; type 2 glycosyltransferase; UDP; uridine 5′-diphosphate
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwj104

Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme's surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.